Supplementation of the human diet with prebiotic substances such as inulin and nondigestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health bene®ts. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bi®dobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bi®dobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bi®dogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential bene®cial effects of inulin metabolism included minor reductions in â-glucosidase and â-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occur principally in the proximal colon.